Flexible methods are needed in the medical field that allow selective activation of various molecular agents for a variety of therapeutic applications, including photodynamic therapy and localized surgical procedures. Desired improvements include enhancements in spatial or temporal control over the location and depth of activation, reduction in undesirable activation and damage to other co-located or proximal agents, tissues or structures, and increased preference in the activation of desired agents over that of other, non-targeted agents.
Since its discovery in the early 1960's, the laser has been anticipated as being capable of providing such selectivity and flexibility. For instance, various linear and non-linear photochemical and photophysical methods useful for harnessing the special capabilities of laser radiation have been developed that provide some improvements for some applications. A typical review of the many uses for lasers in medicine (see e.g. Miller, Biophotonics Intern. September/October (1997) 50–51; Boulnois, Lasers Med. Sci. 1 (1986) 47–66) demonstrates that lasers with optical outputs ranging from continuous wave (CW) to ultrashort pulsed beams and spanning wavelengths from the ultraviolet (UV) and visible to the near infrared (NIR) and infrared (IR) find useful application in medical treatments ranging from elimination of superficial skin disorders to delicate retinal surgery (e.g. Fisher et al., Lasers Surg. Med. 17 (1995) 2–31; Mourou et al., U.S. Pat. No. 5,656,186). Moreover, improved understanding of the non-specific damage properties of laser energy in various tissues (e.g. Hammer et al., IEEE J. Quant. Electron. 32 (1996) 670–678) has allowed practitioners to devise safer and more efficacious uses for the latest laser sources. However, in general, the performance and flexibility of current therapeutic laser methods is less than desired. Specifically, improved photo-activation methods are needed that may be used to selectively effect various therapeutic processes while providing improved performance and flexibility in the application of these processes.
Application of optical radiation to probe or transform molecular agents has been known for many years. For example, linear single-photon optical excitation has been used extensively for activation of molecular therapeutic agents in photodynamic therapy (PDT). (e.g. Fisher et al., Lasers Surg. Med. 17 (1995) 2–31). A generalized Jablonski diagram for such activation is shown in FIG. 1(a), wherein single-photon excitation (10) occurs when a photo-active agent is excited from a lower quantum-mechanically allowed state Sm to a higher quantum-mechanically allowed state Sn upon absorption of a certain energy E1 which is provided by interaction of a single photon P1 with the agent. Typically, intersystem crossing, IX, subsequently occurs to bring the excited agent to a long-lived activated state Tm from which a photochemical reaction R1 can occur. In the case of photodynamic therapy R1 can include production of cytotoxic species, such as singlet oxygen. Unfortunately, performance of such excitation methods has not been as successful as desired. For example, there is strong evidence (see Young, J. Photochem. Photobiol. B, 6 (1990) 237–247) that the optical radiation P1 used in some common treatment regimes can itself produce disease and other undesirable side effects. Furthermore, a less than desirable penetration depth has plagued most efforts at linear optical excitation of molecular therapeutic agents, primarily as a consequence of the effect of optical scatter and absorbance of the UV or visible activating optical radiation.
Various multi-photon optical excitation methods have also been employed in a number of laboratory applications in an effort to achieve specific improvements in the selectivity of photo-activation for certain applications, and to address many of the limitations posed by single-photon excitation. Excitation sources ranging from single-mode, continuous wave lasers to pulsed Q-switched lasers having peak powers in excess of 1 GW have been used in these methods. Most efforts have been aimed at using multi-photon excitation as a means for spectroscopically probing excited state properties or for detecting analytes present in strongly absorbing matrices, such as environmental samples. For example, simultaneous two-photon excitation has been used as a means for stimulating fluorescence emission from molecules present in optically dense media (see Wirth and Lytle, Anal. Chem. 49 (1977) 2054–2057; Fisher et al., Appl. Spectrosc. 51 (1997) 218–226). A generalized mechanism for such activation is shown in FIG. 1(b), wherein simultaneous two-photon excitation (12) occurs when a photo-active agent is excited upon absorption of a certain energy E1 that is provided by the simultaneous, combined interaction of two photons P1′ and P2′ with the agent. Note that if the energies of both photons P1′ and P2′ are identical, the excitation process is termed “degenerate”. The simultaneous interaction of the two photons is frequently described as being mediated by a transient virtual state V with a lifetime on the order of 10 femtoseconds (fs) or less. If both photons do not interact with the agent during this life time, excitation does not occur and the agent fails to reach Sn. Once the agent has been promoted to the higher quantum-mechanically allowed state Sn its photochemical and photophysical properties will be identical to those resulting from single-photon excitation (10). Two-photon excitation has been described for use in microscopy (e.g. Denk et al., U.S. Pat. No. 5,034,613) and as a probe of membrane properties (e.g. Chen and Van Der Meer, Biophys. J. 64 (1993) 1567–1575). The vast majority of this work appears to have been performed using a single, pulsed excitation source such that the two photons interacting with the molecule are of the same wavelength (the degenerate excitation case). However, non-degenerate (two-color) simultaneous two-photon excitation has also been demonstrated (e.g. Lakowicz et al., Photochem. Photobiol. 64 (1996) 632–635).
Far less commonly, simultaneous three-photon excitation has been described to probe the spectroscopy of molecules such as ammonia (e.g. Nieman and Colson, J. Chem. Phys. 68 (1978) 5656–5657), benzene (e.g. Johnson, J. Chem. Phys. 64 (1976) 4143–4148; Johnson and Korenowsk, Chem. Phys. Lett. 97 (1983) 53–56; Grubb et al., J. Chem. Phys. 81 (1984) 5255–5265; Cable and Albrecht, J. Chem. Phys. 85 (1986) 3155–3164), butadiene (e.g. Johnson, J. Chem. Phys. 64 (1976) 4638–4644), and nitric oxide (e.g. Seaver et al., J. Chem. Phys. 87 (1983) 2226–2231), while simultaneous four-photon spectroscopy has seen even more limited application in studies of molecules such as NO2 (e.g. Rockney et al., J. Chem. Phys. 78 (1983) 7124–7131) and butadiene (e.g. McDiarmid and Auerbach, Chem. Phys. Lett. 76 (1980) 520–524). The general theory of such multi-photon spectroscopies (number of photons≦4) has previously been described (see Andrews and Ghoul, J. Chem. Phys. 75 (1981) 530–538). Recently, Lakowicz and co-workers have described the use of degenerate, simultaneous three-photon excitation to study the properties of various fluorophors in the condensed phase (see Gryczynski et al., Biophys. J. 71 (1996) 3448–3453; Lakowicz et al., Biophys. J. 72 (1997) 567–578) and as a possible imaging means for microscopy (see Gryczynski et al., Photochem. Photobiol. 62 (1995) 804–808; Szmacinski et al., Biophys. J. 70 (1996) 547–555).
Additional work using multi-photon excitation has sought to elucidate the physical and chemical properties of agents using complex multi-photon excitation methods (see Xing et al., J. Chem. Phys. 194 (1996) 826–831; Wang et al., J. Chem. Phys. 105 (1996) 2992–2997) or to exert quantum control over excited-state reaction pathways using one or more temporally- or spectrally-tailored laser pulses (see Flam, Science 266 (1994) 215–217; Bardeen et al., Chem. Phys. Lett. 280 (1997) 151–158; Service, Science 279 (1998) 1847–1848; Zare, Science 279 (1998) 1875–1879; Clary, Science 279 (1998) 1879–1882). These reported multi-photon methods, however, generally require staged, sequential application of light energy over periods far in excess of 10 fs in order to allow intra-molecular reorganization to occur. FIG. 1(c) shows a generalized representation of such multi-photon activation, wherein 3+2-photon excitation (14) occurs when a photo-active agent is initially excited to a first higher quantum-mechanically allowed state, Sn, upon absorption of a certain energy, E1, that is provided by the simultaneous, combined interaction of three photons P1″, P2″ and P3″ with the agent (this interaction is mediated by two virtual states, V1″ and V2″). Subsequent excitation occurs upon absorption of a certain additional energy E2 that is provided by the interaction of the agent with two additional photons P4″ and P5″ to promote it to a second, higher quantum-mechanically allowed state, Sp. Typically, there exists a short temporal delay between these two steps E1 and E2, and the second excitation event is often mediated by one or more quantum-mechanically allowed energy states, such as So.
In contrast, optically-induced quantum control of chemical reactions occurs when an agent is sequentially shuttled between several intermediate states using one or more laser pulses occurring over time frames consistent with electronic bond transformation, relaxation and intra-molecular energy transfer (see Fisher et al., Appl. Spectrosc. 51 (1997) 218–226; Draumer et al., Science 275 (1997) 54–57; Zhong et al., J. Am. Chem. Soc. 119 (1997) 2305–12306), which are now believed to occur on scales between approximately 100 fs and 1 picosecond (ps). FIG. 1(d) shows a generalized representation of such a multi-photon quantum control process (16), wherein excitation occurs via three steps E1 E2′ and E3′ which result from sequential interaction of three photons P1′″, P2′″ and P3′″ with the agent. In this example, the first photon P1′″ serves to excite the agent to state Sn, while the second photon P2′″ further excites the agent to state Sr. Delivery of photon P3′″ is timed so as to de-excite the agent from Sr to Tq, producing a reactive state (and hence leading to a chemical reaction R1′) which is not directly accessible from Sm. The temporal delays and multiple quantum-mechanically allowed intermediate states typical of both such multi-photon processes shown in FIGS. 1(c)(14) and 1(d)(16) distinguish them from the simultaneous excitation processes shown in FIG. 1(b)(12), which are essentially single step excitation processes.
Application of quantum control and other specialized excitation methods by Draumer, Zhong, Bardeen and others has lead to the elucidation of two temporal regimes for chemical reactions: (a) a fast regime occurring on the sub-ps time frame, involving intra-molecular electronic transformations; and (b) slow regime occurring on supra-ps time frames, involving intra-molecular reorganization, bond cleavage and inter-molecular interactions. However, events that can be caused to occur in the fast regime can be dramatically different than those possible in the slow regime, since in general the characteristic time constant for fast processes, such as electronic excitation τex, is much shorter than that for relaxation τrelax, such as thermal transfer. This implies that if suitably fast excitation processes are used, excitation can be effected, and the resultant effects completed (such as intra-molecular electronic transformations) before significant relaxation processes (such as bond cleavage or thermal transfer) can occur. Such insight has recently begun to see limited application in the fields of photodynamic therapy and laser ablative surgery. Excitation for durations significantly longer than that necessary for interaction in the fast regime (i.e. for ps- to μs-durations) has been found to support photo-activation pathways that are competitive with the desired photo-activation. However, over nanosecond-time-scales, for example, an excited photodynamic agent may absorb additional photons resulting in an undesirable photochemical transformation. This can render agents completely useless for the desired purpose, by transforming a photodynamic therapy agent into a long-lived, systemically-toxic substance (e.g. Shea et al., J. Biol. Chem. 265 (1990) 5977–5982). Furthermore, excitation of events in the slow regime tends to allow energy to leak into surrounding bonds or media which has previously precluded successful quantum control of reactions (due to energy leakage away from desired bonds) and resulted in collateral tissue damage in ablative laser surgery (due to energy leakage into surrounding tissue).
The advent of lasers capable of routinely producing ultrashort pulses (pulse width≦10 ps), such as the mode-locked titanium:sapphire laser, allows excitation to be substantially limited to the fast regime, vastly improving efficiency of energy delivery to desired treatment targets. The brevity of such pulses substantially precludes competition from alternate photo-activation and relaxation pathways, and should thereby yield controlled activation via only desired mechanisms. For example, Boxer and co-workers (Oh et al., Photochem. Photobiol. 65 (1997) 91–95) and Wachter and co-workers (Fisher et al., Photochem. Photobiol. 66 (1997) 141–155) have recently reported simultaneous two-photon excitation of photodynamic therapy agents, the former demonstrating two-photon spectroscopic properties of psoralen-based agents and the latter demonstrating a two-photon excited photodynamic effect for related agents. In both reports, the use of ultrashort excitation pulses limited photo-activation to the desired mechanism, clearly avoiding competing mechanisms.
Furthermore, Mourou et al. (U.S. Pat. No. 5,656,186) and others attempting to use ultrashort laser pulses to achieve laser-induced breakdown for ablative laser surgery (for example Birngruber et al., IEEE J. Quant. Electron. 23 (1987) 1836–1844; Stern et al., Arch. Ophthal. 107 (1989) 587–592; Watanabe et al., Photochem. Photobiol. 53 (1991) 757–762; Frederikson et al., Arch. Derm. 129 (1993) 989–993; Zair et al., U.S. Pat. No. 5,618,285) have found that the use of pulses with durations less than 10 ps yield substantially finer treatment margins than those achieved using longer pulses as a consequence of the enhanced localization of such effects to the site of optical excitation. Mourou describes laser-induced breakdown as being initiated by multi-photon ionization (18), which is purported to occur as a consequence of non-resonant interaction of NIR light with the aqueous tissue matrix, as shown in FIG. 1(e). Here, the concerted interaction of multiple photons Pm–Pq rapidly promotes water through multiple virtual intermediate states Vm–Vp until ionization occurs (at molecular energies greater than or equal to Sip, the ionization potential for water). This process is termed non-resonant because no allowed first-order (single-photon) or multiple-order (two- or more photon) transitions are intentionally accessed (for example, to maximize energy absorption or excitation efficiency).
Surprisingly then, despite the considerable body of theoretical and experimental work with multi-photon spectroscopy and the widespread availability of ultrashort pulsed sources, the inventors are aware of no reports of the general application of ultrashort pulsed, multi-photon methods for therapeutic applications involving selective activation of endogenous (naturally present) or exogenous (externally supplied) molecular agents to produce an enhanced photodynamic or photophysical (ablative) outcome. As explained supra, the work reported to date has been limited solely to two-photon methods or non-specific multi-photon ablation based on non-resonant laser-induced dielectric breakdown.
Thus, while the substantial body of prior work exemplified by works cited herein clearly demonstrates many attractive features of multi-photon photo-activation and the use of ultrashort pulsed sources for effecting such photo-activation, this work has failed to achieve selective photo-activation of one or more molecular agents with a high degree of spatial control and efficiency to meet the diverse needs of the medical field. Specifically, there appears to be no teaching of the use of or practical methods for effecting this control on target agents and materials and on physical scales that are significant for therapeutic applications.
Therefore, it is an object of the present invention to provide flexible and versatile methods for the treatment of plant or animal tissue with a high degree of spacial selectivity.
It is a further object of the present invention to provide such methods using multi-photon optical excitation methods.
It is another object of the present invention to provide such methods using light from a single pulsed light source and endogenous or exogenous photo-active agents to enhance the spatial selectivity and improve the efficiency of such treatment.
It is yet another object of the present invention to provide such methods using wavelengths of light which are in general less harmful to the plant or animal tissue than the wavelengths of light currently used for the treatment of plant or animal tissue.
It is yet another object of the present invention to provide such methods using light which is less prone to scatter in and be absorbed by plant or animal tissue than the wavelengths of light currently used for the treatment of plant or animal tissue.
Consideration of the specification, including the several figures and examples to follow, will enable one skilled in the art to determine additional objects and advantages of the present invention.